Activation & Proliferation of Hematopoietic Stem Cells

描述（由申请人提供）：造血干细胞（HSC）与胚胎干细胞不同，具有有限的分裂潜力。在再生障碍性贫血和慢性髓性白血病等血液学疾病中，造血干细胞复制潜力的限制似乎很重要。这些限制可能进一步阻碍新的治疗策略的发展，包括体外干细胞扩增和基因治疗。基于这些考虑，可能有助于定义和扩展HSC的复制潜力的研究是重要的和普遍的兴趣。先前由该基金资助的纯化人类“候选”HSC的研究表明，HSC的功能特性在个体发育过程中发生了巨大变化，并且随着年龄的增长，HSC增殖潜力的丧失与可测量的端粒缩短有关。在此，我们建议进一步研究端粒酶和端粒在造血中的作用。具体来说，我们想要验证HSC的复制历史可以通过研究它们的端粒长度来追踪的假设。为了验证这一假设，我们将使用我们实验室开发的精细流式细胞术技术，检测纯化HSC亚群及其培养后代相对于定义的更成熟的细胞群的端粒长度。这些技术还将用于进一步研究正常个体、各种血液学疾病患者和基因型正常狒狒家系的有核血细胞中端粒长度的年龄相关下降。我们将进一步尝试利用基因转移和蛋白质转导策略来操纵HSC的端粒长度，并在体外和体内研究端粒延长的细胞的功能特性。具体目的是：1)研究正常人和各种血液病患者纯化的HSC和有核血细胞治疗前后的端粒长度。2)研究狒狒有核血细胞端粒长度与年龄和基因型的关系。3)研究人工端粒延长和端粒酶抑制对体外和体内纯化“候选”HSC增殖、分化和复制潜能的影响。综上所述，这些研究将为端粒在HSC生物学中的作用提供关键的基线信息。这些信息对于造血的基本理解以及干细胞在血液学内外的应用是相关的。

DESCRIPTION (provided by applicant): Hematopoietic stern cells (HSC), unlike e.g. embryonic stem cells, have a finite potential to divide. Limitations in the replication potential of HSC appear to be important in hematological disorders such as aplastic anemia and chronic myeloid leukemia. Such limitations could furthermore hamper the development of novel therapeutic strategies, including ex vivo stem cell expansion and gene therapy. Based on these considerations, studies that may help define and extend the replicative potential of HSC are important and a general interest. Previous studies with purified human "candidate" HSC funded by this grant have shown that the functional properties of HSC change dramatically during ontogeny and that the loss in HSC proliferative potential with age correlates with measurable shortening of telomeres. Here we propose to further examine the role of telomerase and telomeres in hematopoiesis. Specifically, we want to test the hypothesis that the replication history of HSC can be traced by studies of their telomere length. In order to test this hypothesis, we will examine the telomere length in subsets of purified HSC and their cultured progeny relative to defined populations of more mature cells using refined flow cytometry techniques developed in our laboratory. These techniques will also be used to further study the age related decline in telomere length in nucleated blood cells from normal individuals, patients with various hematological disorders and pedigrees of genotyped normal baboons. We will furthermore attempt to manipulate the telomere length in HSC using gene transfer and protein transduction strategies and study the functional properties of cells with extended telomeres in vitro and in vivo. The specific aims are:1) To study the telomere length in purified HSC and nucleated blood cells from normal individuals and patients with various hematological disorders before and after therapy.2) To study the telomere length in nucleated blood cells from baboons in relation to their age and genotype.3) To study the effect of artificial telomere elongation and telomerase inhibition on the proliferation, differentiation and replicative potential of purified "candidate" HSC in vitro and in vivo.Taken together, these studies will provide crucial baseline information on the role of telomeres in the biology of HSC. Such information is relevant for a basic understanding of hematopoiesis as well as applications of stem cells in and outside hematology.